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WO2002038811A2 - Nouveau dosage permettant de detecter et de quantifier la semi-methylation - Google Patents

Nouveau dosage permettant de detecter et de quantifier la semi-methylation Download PDF

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WO2002038811A2
WO2002038811A2 PCT/US2001/047141 US0147141W WO0238811A2 WO 2002038811 A2 WO2002038811 A2 WO 2002038811A2 US 0147141 W US0147141 W US 0147141W WO 0238811 A2 WO0238811 A2 WO 0238811A2
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methylation
dna
cpg
methylated
cells
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WO2002038811A3 (fr
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Peter A. Jones
Gangning Liang
Yashitaka Tomigahara
Peter W. Laird
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University of Southern California USC
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Priority to EP01984994A priority patent/EP1379685A2/fr
Priority to JP2002541125A priority patent/JP2004535757A/ja
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • Patent Application Serial Number 60/247,191 entitled “A NEW ASSAY FOR THE DETECTION AND QUANTITATION OF HEMIMETHYLATION,” filed on 08 November 2000.
  • the present invention relates to DNA methylation, and in particular hemi- methylation.
  • the present invention provides a method to measure the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a particular DNA sequence, in a pool of DNA molecules having mixed DNA methylation states at said CpG dinucleotide.
  • the present invention also provides for methods to calculate the rates of de novo-, and maintenance-methylation per cell division that occur at said CpG dinucleotide.
  • DNA is methylated only at cytosines located 5' to guanosine in the CpG dinucleotide. This modification has important regulatory effects on gene expression predominantly when it involves CpG rich areas (CpG islands) located in the promoter region of a gene sequence. Extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X chromosome of females. Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells and has been frequently associated with transcriptional inactivation of tumor suppressor genes in human cancers.
  • DNA methylases transfer methyl groups from a universal methyl donor, such as S- adenosyl methionine, to specific sites on the DNA.
  • Mammalian cells possess methylases that methylate cytosine residues on DNA that are 5' neighbors of guanine in CpG dinucleotides (CpG) . This methylation may play a role in gene inactivation, cell differentiation, tumorigenesis, X-chromosome inactivation, and genomic imprinting. CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation and parental specific imprinting where methylation of 5' regulatory regions can lead to transcriptional repression.
  • CpG dinucleotides CpG dinucleotides
  • DNA methylation is also a mechanism for modifying the base sequence of DNA without altering its coding function.
  • DNA methylation is a heritable, reversible and epigenetic change. Yet, DNA methylation has the potential to alter gene expression, which has profound developmental and genetic consequences.
  • DNA methylation is required for mammalian development (Li et al, 1992; Okano et al., 1999) yet the mechanisms responsible for the establishment and copying of methylation patterns remain almost completely unknown.
  • 5-Methylcytosine is asymmetrically distributed in the genome and is most commonly found in CpG-poor regions, since most CpG islands in somatic cells remain methylation-free, except for the promoters of imprinted genes and genes on the inactive X-chromosome (Bird et al., 1985).
  • Methylation occurs after cytosine has been incorporated into DNA in a process catalyzed by DNA methyltransferases ("Dnmts") which transfer the methyl group from S- adenosylmethionine to the 5 '-position of the pyrimidine ring in, characteristically but not exclusively, the context of the palindromic CpG dinucleotide (Ramsahoye et al., 2000).
  • Dnmts DNA methyltransferases
  • Three Dnmt enzymes are known in mouse and human, and these have overlapping yet distinct abilities to methylate "hemimethylated” and completely unmethylated CpG dinucleotide pairs.
  • Hemi-methylation is defined as a state in which the two opposing cytosines on either DNA strand in a single palindromic CpG dinucleotide differ in that one is methylated at the C-5 position, and the other is not.
  • Dnmtl The predominant Dnmt in the cell, Dnmtl, was cloned and characterized by Bestor and colleagues (1988) and is localized to replication machines in the S-phase nucleus (Leonhardt et al., 1992; Rountree et al., 2000). Since Dnmtl shows a preference for hemimethylated CpG pairs (Gruenbaum et al., 1981; Bestor and Ingram, 1983), it is considered to be an excellent candidate for copying the pattern of methylation present on the parental strand after DNA has been replicated. However, Dnmtl is capable of modifying unmethylated DNA in the test tube, and is thus also a candidate for inducing de novo methylation.
  • Dnmts, Dnmt3a and 3b show equal activities in vitro for unmethylated and hemimethylated substrates (Okano et al., 1998), and have been shown to be capable of de novo methylation of transfected DNA in culture (Hsieh, 1999) and in Drosophila (Lyko et al, 1999).
  • satellite DNAs appear to be a preferred target for the human DNMT3B enzyme, because these satellite DNA sequences are specifically undermethylated in patients with ICF syndrome, characterized by germ-line mutations in the DNMT3B gene (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999).
  • the present invention provides a method to measure the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a pool of DNA with mixed DNA methylation states at that CpG dinucleotide, comprising: extracting DNA from the various cell types; digesting the DNA sample with an excess of a methylation-sensitive restriction endonuclease that cleaves according to a recognition sequence motif comprising a palindromic CpG methylation site, whereby the endonuclease cleaves the DNA at a specific recognition motif position if the methylation state of the respective palindromic CpG methylation site is un-methylated, but not hemi-methylated or fully-methylated (e.g., Hpall); treating the DNA with bisulfite; amplifying the treated DNA by PCR with primers flanking the specific recognition motif position (e.g., the Hpall site); and determining the degree of hemi-
  • analysis of individual cloned PCR products for the presence of C or T at the first position of the specific CpG dinucleotide was accomplished using a methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay (Gonzalgo and Jones, 1997).
  • Ms-SNuPE methylation-sensitive single nucleotide primer extension
  • the present invention further provides a novel set of equations to calculate the rate of de novo methylation (n) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a Hpall site.
  • the present invention further provides a hemimethylation detection kit useful for measuring the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a pool of DNA with mixed DNA methylation states at that CpG.
  • the present invention further provides a hemimethylation detection kit useful for measuring the rate of de novo methylation (n) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a palindromic CpG methylation site.
  • Figure 1A shows the patterns of methylation in ES Cells.
  • the methylation status of the four CpG sites in individual molecules of DNA (Fragment Cl-f) comprising the 440 bp sequence were assessed by cloning of individual molecules followed by MS-SNuPE analysis of the four sites as indicated.
  • Figure 2 shows the detection and quantitation of hemi-methylation in individual cloned DNA molecules according to the present invention.
  • the experimental approach used to detect hemi-methylation in the ES cells comprises precutting genomic DNA with Hpall, followed by bisulfite treatment, then cloning individual PCR products, and assessing the methylation status of, e.g., site 2 by MS-SNuPE analysis.
  • Figure 3 shows the measured steady state methylation levels, methylation rates, and an overview of the procedure used to calculate de novo (n) and maintenance (m) methylation rates (expressed as the fraction of substrate molecules converted per cell division) at a single CpG dinucleotide within a Hpall site, starting with the measurement of two experimental variables ("P" and "S").
  • Figure 4A summarizes results from such pulse-chase experiments which show that DNA synthesized during a 1 hr pulse of BrdU in wild type M1/3A/3B cells was not methylated to its final level until 24-48 hr after synthesis.
  • Figure 4B shows that the delayed methylation of DNA requires active DNA methyltransferase enzymes.
  • Figure 5 shows the maturation of methylation pattern cells. The methylation status of individual DNA molecules was determined immediately after a 1 hr pulse with BrdU, or after a chase period (24 r) in medium not containing BrdU.
  • Figures 6A,B and C illustrate the proposed model for the interactivity between DNA methyltransferase enzymes in ES cells.
  • Figure 6A depicts how methylation patterns are maintained in wild-type cells containing all three DNA methyltransferase enzymes.
  • Figure 6B indicates how methylation is proposed to occur in cells lacking both Dnmt3a and Dnmt3b enzymes.
  • Figure 6C shows the situation in M3A/3B cells, which lack Dnmtl. A mosaic methylation pattern is present with patches of methylation and unmethylation and a substantial fraction of hemi-methylated sites.
  • Figure 7A shows methylation sensitive fingerprints of M1/3A/3B, M3A/3B, and Ml ES cell DNA after enzyme digestion with each of Rsal, Rsal with Hpall, or Rsal with Mspl for 16 h. Bands which appear to be hypomethylated only in lane 2 are indicated by solid arrowheads (Class I). Bands which appear to be hypomethylated in both lanes 2 and 3 are indicated by open arrowheads (Class II). Functional DNA methyltransferase genes are indicated by solid squares and inactivated genes are indicated by open squares. GCP1, 2, 4 are GC poor primers.
  • Figure 7B shows a Southern blot analysis of genomic DNA from M1/3A/3B,
  • Figure 7C shows a Ms-SNuPE analysis for fragment Cll-d from M1/3A/3B, M3A/3B, and Ml ES cell DNA. Quantitative methylation analysis of three CpG sites in Cll-d. The percent methylation represents the average of three site using the following equation on Phospholmager quantitation (methylated)C/[(methylated)C + (unmethylated)T] X 100. CpG sites are represented by tick marks; striped boxes represent repetitive sequences; arrows represent PCR primers.
  • Figure 8 shows the sequence properties of hypomethylated fragments isolated from methylation sensitive fingerprints in ES cells (Figure 7A CpG sites are represented by tick marks; striped boxes represent repetitive sequences; upward arrows represent Hpall sites. indicates bands that are not included in Figure 7A.
  • Figure 9 shows the patterns of Methylation in ES Cells at Different Regions.
  • the methylation status of individual molecules of DNA from the Cl-f (Class I), CII-d and A- repeat regions (Class II) were assessed by cloning of individual bisulfite converted molecules followed by Ms-SNuPE or automated sequencing.
  • CpG sites are represented by tick marks.
  • Methylation status of CpG dinucleotides (black wedges/circles) methylated; (white wedge/circles) unmethylated.
  • Horizontal rows of wedges/circles indicate individual molecules that were sequenced after PCR amplification and cloning of bisulfite-treated DNA.
  • the striped region indicated repeat sequences.
  • Figure 10A shows the distribution of methylation status at Cl-f and CII-d regions, and an overview of the procedure used to calculate distribution of methylation status (fully methylated, hemimethylated, and fully unmethylated) at a single CpG dinucleotide within a Hpall site, starting with the measurement of two experimental variables ("P" and "S"). A description of each variable is given in the first column, along with the equation used to derive the calculated variables (see Experimental Procedures). The second column illustrates the subset of methylation states representing that variable (white box), as well as all the other methylation states being assessed in that particular step (gray box).
  • Figure 11 shows the recovery of methylation after 5-Aza-CdR treatment.
  • the indicated cell types were treated for 24 h with 3x10 "7 M 5-aza-CdR and medium changed and the cells propagated further.
  • DNA was extracted at the times indicated after the drug was added to the cultures and the methylation status of the Cl-f (black) and CII-d fragments
  • Figure 12 shows the methylation kinetics of newly synthesized DNA at Cl-f and C ⁇ - d.
  • ES cells containing the genes for the indicated DNA methyltransferases were pulsed for 1 hr with BrdU. The pulse was then removed and fresh medium added to the cells during the chase period. The DNA was extracted from the cells at various times after the pulse began and immunoprecipitated to isolate the BrdU containing DNA.
  • the methylation status of Hpall sites in Cl-f and CII-d at the indicated time points were determined by quantitative MS-SNuPE analysis. The data represent the mean values in two to six experiments and the error bars indicate the standard deviations.
  • GC Content refers, within a particular DNA sequence, to the [(number of C bases + number of G bases) / band length for each fragment].
  • CpG Island refers to a contiguous region of genomic DNA that satisfies the criteria of (1) having a frequency of CpG dinucleotides corresponding to an 'Observed/Expected Ratio" >0.6), and (2) having a "GC Content” >0.5.
  • CpG islands are typically, but not always, between about 0.2 to about 1 kb in length.
  • “Hpall” refers to the art-recognized restriction endonuclease having the palindromic “CCGG” (top strand): “GGCC” (bottom strand) recognition motif that comprises two CpG dinucleotide sequences (one on each DNA strand) that function as potential methylation sites.
  • Rsal refers to the art-recognized restriction endonuclease having the "GTAC” (top strand): “CATG” (bottom strand) recognition motif.
  • Excess of a methylation-sensitive restriction endonuclease used to digest DNA refers to an amount sufficient, under the reaction conditions, to bring about essentially complete digestion of DNA, before bisulfite treatment to ensure accuracy.
  • Methods refers to the presence or absence of 5-methylcytosine ("5- mCyt") at one or a plurality of CpG dinucleotides within a DNA sequence.
  • Methylation states at one or more particular palindromic CpG methylation sites (each having two CpG CpG dinucleotide sequences) within such a DNA sequence include "unmethylated,” “fully- methylated” and "hemimethylated.”
  • Hemimethylation refers to the methylation state of a palindromic CpG methylation site, where only a single cytosine in one of the two CpG dinucleotide sequences of the palindromic CpG methylation site is methylated (e.g., 5'-CC M GG-3' (top strand): 3'-GGCC- 5' (bottom strand)).
  • “Hypermethylation” refers to the methylation state corresponding to an increased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
  • Hypomethylation refers to the methylation state corresponding to a decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
  • De Novo methylation refers to the conversion of unmethylated post-synthesis CpG dinucleotide sequences (within a palindromic CpG methylation site) to fully methylated CpG sequences.
  • Maintenance methylation refers to the conversion of post-synthesis hemi- methylated CpG dinucleotide sequences (within a palindromic CpG methylation site) to fully methylated CpG sequences.
  • Methods refers to any assay for determining the methylation state of a CpG dinucleotide within a sequence of DNA.
  • MS.AP-PCR Methods of PCR (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction) refers to the art-recognized technology that allows for a global scan of the genome using CG-rich primers to focus on the regions most likely to contain CpG dinucleotides, and described by Gonzalgo et al., Cancer Research 57:594-599, 1997.
  • Methods-Light refers to the art-recognized fluorescence-based real-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999.
  • Ms-SNuPE Metal-sensitive Single Nucleotide Primer Extension
  • MSP Metal-specific PCR
  • COBRA combined Bisulfite Restriction Analysis
  • MCA Metal CpG Island Amplification
  • DGE art-recognized 2-dimensional gel electrophoresis (2-DGE) methods.
  • the present invention provides a novel and straightforward technique to quantitate hemimethylation levels. Prior to the development of this method, there had been no accurate way to determine hemimethylation levels at specific CpG dinucleotides in the genome.
  • the steady-state levels of hemimethylation and full methylation at a CpG dinucleotide provide sufficient information to allow calculation of the rates of de novo methylation and of maintenance methylation.
  • the present invention provides a unique set of equations that reflect this concept. This led to the discovery that cells containing only the Dnmt3a and 3b enzymes show significantly more maintenance methyltransferase activity at this site than de novo methyltransferase activity. This contrasts with the prior view of these enzymes as predominantly de novo methyltransferases, but supports our proposed concept of post-Dnmtl repair maintenance methyltransferases.
  • the present invention provides provides a method to measure the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a particular DNA sequence, in a pool of DNA molecules having mixed DNA methylation states at said CpG dinucleotide.
  • the present invention also provides for methods to calculate the rates of de novo-, and maintenance-methylation per cell division that occur at said CpG dinucleotide.
  • the region selected for study was part of the transcribed region of a gene expressed in mouse mammary and thymus tissue. It was representative of the situation in most of the mammalian genome in which CpG islands, frequently located in promoters, are methylation free and most of 5-methylcytosine is found in coding regions and repetitive elements (Bird et al., 1985; Yoder et al., 1997; Jones, 1999). Because of the complexities of dealing with at least three enzymes with overlapping activities, the patterns and kinetics of methylation in wild-type cells were compared with cells containing either a homozygous mutation of the Dnmtl gene or with a double Dnmt3a/3b knockout.
  • Pulse chase experiments showed that, contrary to current assumptions, DNA was not methylated to its final level immediately after synthesis, but rather that methylation occurred in two phases.
  • the data are most consistent with a model whereby Dnmtl acts soon after replication but leaves gaps of hemimethylated DNA which are then filled in by Dnmt3a and/or 3b, which therefore have roles as "methylation repair" enzymes in addition to their potential roles as de novo methyltransferases.
  • ⁇ S cells were seeded at 9 X 10 5 cells per 60 mm dish with feeder cells 72 hr before being pulsed for 1 hr in 2.5 ml medium containing 1 X 10 "4 M BrdU (Sigma).
  • the BrdU-containing medium was then removed, the cells washed once with regular medium at 37°C then chased with 4 ml medium supplemented with 1 X 10 "4 M thymidine (Sigma).
  • the BrdU was also chased with 10 "6 M 5-Aza-CdR (Sigma), 0.1 ng/ml Trichostatin (TSA) (Sigma), 5 X 10 "6 M Aphidicolin (Sigma), or 50 ng/ml Colcemid (Sigma).
  • Genomic DNA was obtained following lysis with 100 mM NaCl, 10 mM ⁇ DTA, 1% SDS, and 1 ⁇ g/ml Proteinase K and purified by phenol and chloroform extractions and ethanol precipitation. The yield and purity of the DNA was determined by optical density measurement and 300 ⁇ g of DNA digested with 3000 units of Rsal (Roche) for 16 hr at 37°C. Digested DNA was purified again by phenol and chloroform extractions, ethanol precipitated, and resuspended in 500 ⁇ l T ⁇ buffer pH 7.5 with 50 ⁇ l 10X immunoprecipitation buffer (sodium phosphate (pH 7.0)), 0.14 M NaCl, 0.05% Triton X- 100).
  • the samples were denatured at 95°C for 5 min, cooled on ice for 2 min, and immediately mixed with 2.4 ⁇ l anti-BrdU antibody (25 ⁇ g/ml) per ⁇ g DNA (Becton Dickinson) (Rideout et al., 1994). After 30 min incubation at room temperature with mild rocking, 0.46 ⁇ l anti-mouse IgG rabbit (2.6 mg/ml) per ⁇ g DNA (Sigma) was added and incubation continued for another 30 min at room temperature.
  • the precipitate was collected after 5 min centrifugation at 13,000 rpm in a cold microcentrifuge, dissolved in 200 ⁇ l T ⁇ pH 7.5 and treated with Proteinase K at 50°C for more than 12 hr. DNA was deproteinized by phenol and chloroform extractions and ethanol precipitated.
  • Ms-SNuPE The Ms-SNuP ⁇ technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest.
  • Typical reagents for Ms-SNuPE analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for specific gene; reaction buffer (for the Ms-SNuPE reaction); and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • Methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assays The mean cytosine methylation levels of CpG sites 2, 3 in the fragment were determined by treatment of DNA (2 ⁇ g) with sodium bisulfite according to Frommer et al. (1992).
  • Methylation analysis was performed using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay (Gonzalgo and Jones, 1997). For Examples 1-5. below:
  • the sequences of the primers used for bisulfite-treated DNA amplification were as follows: Top strand: 5' primer (5'-AATGTGTAATATTTTTATGGTTTTTTTAGAATGG- 3') (SEQ ID NO:l), 3' primer (5'-TTACAAAAAAAATACCTCTTCCTTACTAAAC-3') (SEQ ID NO:2). Bottom strand: 5' primer (5'-TGGTATTTGGAAAGATTGTAGGAAAG- 3') (SEQ ID NO:3) 3' primer (5'-CAATACCTCTATAACCCTTCCAAA-3') (SEQ ID NO:4).
  • PCR reactions were performed in 25 ⁇ l total volume under the following conditions: 100-200 ng bisulfite treated DNA, 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 100 mM KCl, 0.5 ⁇ M final concentration of each primer, 200 ⁇ M each of the four deoxynucleotide triphosphates, and 0.5 U Taq polymerase (Sigma) complexed with Taq polymerase antibody (Clontech).
  • DNA was initially denatured at 94°C for 5 min, 94°C for 1 min, 56°C for 50 sec, and 72°C for 1 min for 35 cycles, with a final extension at 72°C for 4 min.
  • PCR products were gel purified with the Qiaquick Gel Extraction Kit (Qiagen), and the template was resuspended in 30 ⁇ l H 2 O.
  • Ms-SNuPE primers for top strand site 2 (5'-AATAATTTTGTTTTTTTGGATATT- 3') (SEQ ID NO:5), site 3 (5'-AAATTTTGTTTTTGGTTGTAAA-3') (SEQ ID NO:6), for bottom strand site 2 (5'-AGGAATAGAATTTGAGATATT-3') (SEQ ID NO:7), site 3 (5'- TAAATTGTTTTAATTAGATTAATAA-3 ') (SEQ ID NO:8).
  • Ms-SNuPE reactions were performed in 10 ⁇ l total volume under the following conditions: 4 ⁇ l Qiaquick product, 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2s 100 mM KCl, 0.5 ⁇ M final concentration of each primer, and 1 ⁇ Ci of either [ 32 P]dCTP or [ 32 P]dTTP.
  • Primer extension conditions were: 95°C for 2 min, 50°C for 2 min, 72°C 1 min. The reactions were combined with 4 ⁇ l stop solution before being denatured at 95°C for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7M urea).
  • PCR primers were designed specifically to amplify only bisulfite converted DNA and control experiments showed non-amplification of unconverted DNA with these primers. Also, sequencing of PCR products after bisulfite treatment showed less than 1% residual Cs at not CpG sites indicating that the assays were valid for methylation status at CpG sites.
  • sequences of the primers used for bisulfite-treated DNA amplification were as follows: C - (Top strand) 5 ' primer, 5 '-AATGTGTAATATTTTTATGGTTTTTTTAGAATGG-3 ' (SEQ ID NO:l), 3' primer, 5'-TTACAAAAAAAATACCTCTTCCTTACTAAAC-3' (SEQ ID NO:2).
  • A-Repeats 5' primer, 5'-TGATTTATTTATTAGAGGTTTTAGG-3' (SEQ ID NO: 19), 3' primer, 3 '-AC ATAAAAAAACAAACTACCC-3' (SEQ ID NO:20).
  • PCR Conditions Cj ⁇ f. 95°C for 2 min, 95°C for 1 min, 56°C for 50 sec, and 72°C for
  • PCR products were gel purified with the Qiaquick Gel Extraction Kit (Qiagen), and the template was resuspended in 30 ⁇ l H 2 O. Ms-SNuPE Primers.
  • Cj ⁇ f 5'-AATAATTTTGTTTTTTTTTGGATATT-3' (SEQ ID NO:5) (Hpall site); CII-d: 5'-TTTTATTTATTGTTATTATGG-3' (SEQ ID NO:21) (site 1), 5'-GGTATAGTTTGAGTAT-3' (site 2) (SEQ ID NO:22), 5- TATTTTTTAATAGTATTATTTTTTAT-3' (SEQ ID NO:23) (site 3, Hpall Site).
  • Ms-SNuPE reactions were performed in 10 ⁇ l total volume under the following conditions: 4 ⁇ l Qiaquick product, 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 100 mM KCl, 0.5 ⁇ M final concentration of each primer, and 1 ⁇ Ci of either [ 32 P]dCTP or [ 32 P]dTTP.
  • Primer extension conditions of Cl-f and CII-d were: 95°C for 1 min, 46°C for 30 sec, 72°C for 20 sec.
  • the reactions were combined with 4 ⁇ l stop solution before being denatured at 95°C for 5 min and loaded onto a 15% denaturing polyacrylamide gel (7M urea). Quantitation of methylation levels was performed on a Molecular Dynamics Phosphorlmager.
  • Bisulfite genomic sequencing by Ms-SnuPE Bisulfite Genomic Sequencing by Ms-SNuPE or Automated DNA Sequencer. Traditionally patterns are determined by the cloning of individual bisulfite treated molecules followed by DNA sequencing. It was more convenient to subject individually cloned molecules to Ms-SNuPE analysis to rapidly assess how the four sites were methylated (Cl-f and CII-d). The PCR products from bisulfite converted DNA were ligated into pCRII cloning vector (Invitrogen, San Diego, CA). Individual plasmid clones were amplified by Ml 3 primers (forward and backward). The PCR product was used to sequence by Ms-SNuPE.
  • A-repeats clones were sequenced by automated DNA sequencer at USC/Norris Comprehensive Cancer Center microchemical facility. 5-Aza-CdR Treatments. Cells were plated (2x10 6 cells/60 mm dish) and treated 24 h later with 3x10 "7 M 5-Aza-CdR (Sigma). The medium was changed 24 h after drug treatment and every subsequent day. DNA was isolated at the indicated days after treatment as described (Pfeifer, et al. P.N.A.S USA 87:8252-8256, 1990).
  • Hemimethylation assay The fact that the enzyme Hpall will not cut the sequence CCGG in either the fully or hemimethylated configuration followed by bisulfite treatment was used to determine whether unmethylated cytosine occurred in the context of a CpG sequence in a Hpall insensitive site.
  • Two ⁇ 4 ⁇ g D ⁇ A were digested by Rsal and Hpall (10 units per ⁇ g) for 16 hr at 37°C and then Hpall added (10 units per ⁇ g) for another 2 hr. The digests were then analyzed for methylation at site 2 by cloning of individual PCR-amplified bisulfite treated molecules as described above.
  • D percent of Hpall-resistant molecules with double-stranded (full) methylation
  • H The amount of hemimethylation
  • F full methylation
  • F DP/(S+D).
  • the basis for these two equations is the assumption that the ratio between hemimethylation and full methylation is constant for a particular D ⁇ A sample. Therefore, this ratio, which can be determined from the measurement of S (but which is lacking information on unmethylated DNA), can be applied to the measurement of all methylation within a single strand (P).
  • Methylation-Sensitive AP-PCR Methylation-sensitive AP-PCR and isolation of fragments of interest were performed as previously described Gonzalgo, et al., Cancer Research 57:594-599, 1997; Liang, et al., Genomics 53:260-268, 1998).
  • the following CpG- poor primers were used for AP-PCR analysis: GCP1, 5'-CACATGGTTCTGC-3' (SEQ ID NO:13); GCP2, 5'-GTCTCTATGACCC-3' (SEQ ID NO:14); GCP4, 5'- CTTACTGTGCCAC-3' (SEQ ID NO: 15).
  • the following pairs were used in the AP-PCR reaction: GCP1/GCP2, GCP2/GCP4, GCP1/GCP4.
  • plasmid insert DNA 32 Approximately 100 ng of plasmid insert DNA were P-labeled by random priming and used to probe the filters. All hybridizations were performed in 500 mM NaPO4 (pH 6.8), 7% SDS, 1 mM EDTA (pH 8.0) at 65°C for 16 hr. Membranes were washed twice at 65°C with 2XSSC/1%SDS followed by three washes with 0.5X SSC/1% SDS at room temperature. They were then exposed to autoradiographic film at -80°C. Quantitation of methylation levels was performed on a Molecular Dynamics Phosphorlmager.
  • Methylation Assay Procedures Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention for sequence determinations. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a DNA sequence. Such assays involve, among other techniques, DNA sequencing of bisulfite-treated DNA, PCR (for sequence-specific amplification), Southern blot analysis, use of methylation-sensitive restriction enzymes, etc.
  • genomic sequencing has been simplified for analysis of DNA methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992).
  • restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used, e.g., the method described by Sadri & Hornsby (Nucl. Acids Res. 24:5058-5059, 1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).
  • COBRA COBRA analysis is a quantitative methylation assay useful for determining
  • DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite- treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by
  • Typical reagents for COBRA analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); restriction enzyme and appropriate buffer; gene-hybridization oligo; control hybridization oligo; kinase labeling kit for oligo probe; and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • assays such as "MethyLight” (a fluorescence-based real-time PCR technique) (Eads et al, Cancer Res. 59:2302-2306, 1999), Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP”; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; US Patent No. 5,786,146), and methylated CpG island amplification ("MCA”;Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with other of these methods.
  • MSP methylation-specific PCR
  • MCA methylated CpG island amplification
  • the MethyLight assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan ®) technology that requires no further manipulations after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight process begins with a mixed sample of genomic DNA that is converted, in a sodium bisulfite reaction, to a mixed pool of methylation-dependent sequence differences according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil).
  • TaqMan ® fluorescence-based real-time PCR
  • Fluorescence-based PCR is then performed either in an "unbiased” (with primers that do not overlap known CpG methylation sites) PCR reaction, or in a “biased” (with PCR primers that overlap known CpG dinucleotides) reaction. Sequence discrimination can occur either at the level of the amplification process or at the level of the fluorescence detection process, or both.
  • the MethyLight may assay be used as a quantitative test for methylation patterns in the genomic DNA sample, wherein sequence discrimination occurs at the level of probe hybridization.
  • the PCR reaction provides for unbiased amplification in the presence of a fluorescent probe that overlaps a particular putative methylation site.
  • An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers, nor the probe overlie any CpG dinucleotides.
  • a qualitative test for genomic methylation is achieved by probing of the biased PCR pool with either control oligonucleotides that do not "cover” known methylation sites (a fluorescence- based version of the "MSP" technique), or with oligonucleotides covering potential methylation sites.
  • the MethyLight process can by used with a "TaqMan®” probe in the amplification process.
  • double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two sets of PCR reactions using TaqMan® probes; e.g., with either biased primers and TaqMan® probe, or unbiased primers and TaqMan® probe.
  • the TaqMan® probe is dual-labeled with fluorescent "reporter” and "quencher” molecules, and is designed to be specific for a relatively high GC content region so that it melts out at about 10 °C higher temperature in the PCR cycle than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing/extension step.
  • Taq polymerase As the ⁇ Taq polymerase enzymatically synthesizes a new strand during PCR, it will eventually reach the annealed TaqMan® probe. The Taq polymerase 5' to 3' endonuclease activity will then displace the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of its now unquenched signal using a real-time fluorescent detection system.
  • Typical reagents for MethyLight analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); TaqMan® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.
  • Ms-SNuPE The Ms-SNuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest.
  • Typical reagents for Ms-SNuPE analysis may include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for specific gene; reaction buffer (for the Ms-SNuPE reaction); and radioactive nucleotides.
  • bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components.
  • MSP methylation-specific PCR
  • DNA is modified by sodium bisulfite converting all unmethylated, but not methylated cytosines to uracil, and subsequently amplified with primers specific for methylated versus unmethylated DNA.
  • MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples.
  • Typical reagents e.g., as might be found in a typical MSP-based kit
  • MCA MCA.
  • the MCA technique is a method that can be used to screen for altered methylation patterns in genomic DNA, and to isolate specific sequences associated with these changes (Toyota et al., Cancer Res.
  • restriction enzymes with different sensitivities to cytosine methylation in their recognition sites are used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that show differential methylation are cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments are then used as probes for Southern analysis to confirm differential methylation of these regions.
  • Typical reagents for MCA analysis may include, but are not limited to: PCR primers for arbitrary priming Genomic DNA; PCR buffers and nucleotides, restriction enzymes and appropriate buffers; gene-hybridization oligos or probes; control hybridization oligos or probes.
  • kits for Detection of Hemimethylated CpG-containing Nucleic Acid provides a hemimethylation detection kit for measuring the fraction of DNA molecules that is hemi-methylated at a specific CpG dinucleotide in a pool of DNA with mixed DNA methylation states at that CpG.
  • the present invention further provides a hemimethylation detection kit useful for measuring the rate of de novo methylation (n) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a Hpall site.
  • the reagents required to perform one or more art-recognized methylation assays are combined with specific primers or probes to determine the hemimethylation state of CpG-containing nucleic acids, or the rate of de novo methylation ( ⁇ ) and the rate of maintenance methylation (m) at a single CpG dinucleotide within a Hpall site, according to the present invention.
  • the Ms-SnuPE methylation assay could be used alone or in combination with other methylation assay methods known in the art, along with specific primers or probes to determine the hemimethylation state of a CpG dinucleotide within a particular genomic sequence.
  • the methylation status of the four CpG sites in individual DNA strands in cells containing all three Dnmts (M1/3A/3B cells) or containing Dnmtl (Ml cells) or Dnmt3a and 3b only (M3A/3B cells) were assessed in detail to investigate potential differences in the patterns of methylation.
  • the nomenclature used to describe the cell types was selected to focus on which enzymes were present in the cells rather than those which > were absent. Minimal differences in methylation levels and patterns were seen in cells lacking either Dnmt 3 a or 3b only. Initially, the methylation patterns at sites 2-4 were determined by cloning of individual bisulfite treated molecules followed by DNA sequencing (top fragments in Fig. IB).
  • Figure 1 A shows the patterns of methylation in ES Cells.
  • the methylation status of the four CpG sites in individual molecules of DNA (Fragment Cl-f) comprising the 440 bp sequence were assessed by cloning of individual molecules followed by MS-SNuPE analysis of the four sites as indicated.
  • Example gels of samples labeled with either dCTP "C” or dTTP "T" are shown.
  • CpG sites in individual molecules are scored as being methylated (filled boxes) or unmethylated as shown (open boxes) so that patterns can be deduced.
  • the data from the upper section within each panel were obtained by direct sequencing of fragments containing sites 2 through 4.
  • the bottom sections are for all four sites.
  • the status of methylation at the sites in individual molecules is read from left to right.
  • Figure IB also shows the average percent of methylation at the four sites in the three cell types examined.
  • Sites 2 and 4 were most methylated on average whereas sites 1 and 3 were less efficient methyl acceptors.
  • the methylation pattern in the M3A/3B cells were, however, completely different in that two classes of molecules were present. The majority of molecules in which all four sites were simultaneously assessed were methylation-free (72%), 14% had only one site methylated but 14% had multiple sites methylated on a single molecule.
  • Figure 2 shows the detection and quantitation of hemi-methylation in individual cloned DNA molecules according to the present invention.
  • the experimental approach used to detect hemi-methylation in the ES cells comprises precutting genomic DNA with Hpall, followed by bisulfite treatment, then cloning individual PCR products, and assessing the methylation status of, e.g., site 2 by MS-SNuPE analysis. Typical results are shown for these MS-SNuPE results in which a signal in the T lanes of example clones indicates hemimethylation with the top strand being unmethylated.
  • the presence of a signal in the "C" lane indicates that the site may be either hemi-methylated (i.e., the bottom strand is unmethylated) or fully methylated.
  • the distribution between these two scenarios is determined by calculation, assuming that there is an equal probability of hemi-methylation of either the top or bottom strand.
  • DNA extracted from the various cell types was digested with an excess of Hpall, bisulfite treated and then amplified by PCR with primers flanking the Hpall site (site 2). Since the Hpall site is located between the primers, molecules without full or hemi- methylation of site 2 would not be amplified and thus be excluded from the analysis.
  • the degree of hemi-methylation at a particular site can be accurately determined by averaging results obtained by analysis of individual cloned PCR products for the presence of C or T at site 2 (for example). Since only the top strand is analyzed in this approach and methylation of either the top or bottom strand is possible, the degree of hemi-methylation was calculated as indicated in "Experimental Procedures," above. These calculations assume that there is an equal probability of hemi-methylation of either the top or bottom strand, a supposition which was supported by additional experiments (see Examples 6-8 below).
  • Figure 3 shows the measured steady state methylation levels, methylation rates, and an overview of the procedure used to calculate de novo (n) and maintenance (m) methylation rates (expressed as the fraction of substrate molecules converted per cell division) at a single CpG dinucleotide within a Hpall site, starting with the measurement of two experimental variables ("P" and "S").
  • P two experimental variables
  • S two experimental variables
  • the unmethylated CpGs are absent in the case of the S and D variables, since the Hpall digestion removes these molecules from consideration at this step.
  • the data obtained for the three cell lines for the measured variables "P" and "S” are shown in the columns on the right as percentages, followed by the absolute numbers of molecules assessed. It is assumed that the top and bottom strand are equal in their methylation levels and rates. On average, differences between the newly synthesized strand and daughter strand after DNA replication should be distributed equally between the top and bottom strand in a large population of cells.
  • Figure 3 also shows the rates of de novo and maintenance methylation which could be calculated from this data (see “Experimental Procedures,” above). Interestingly, Figure 3 shows that M3A/3B cells had higher rates of hemi-methylase activity (0.37) than of de novo methyltransferase activity (0.07). These results indicate that Dnmtl and Dnmt3a/3b acted coordinately to ensure a low proportion of hemi-methylated sites in the wild type cells.
  • Figure 4A summarizes results from such pulse-chase experiments which show that DNA synthesized during a 1 hr pulse of BrdU in wild type M1/3A/3B cells was not methylated to its final level until 24-48 hr after synthesis.
  • ES cells containing the genes for the indicated DNA methyltransferases were pulsed for 1 hr with BrdU. The pulse was then removed and fresh medium added to the cells during the chase period. The DNA was extracted from the cells at various times after the pulse began and immunoprecipitated to isolate the BrdU containing DNA. The methylation status of sites 2 and 3 were averaged at the indicated time points after determination by quantitative MS-SNuPE analysis. The data represent the mean values for the determinations on the top and bottom strands in two to three experiments and the error bars indicate the standard deviations.
  • Figure 4B shows that the delayed methylation of DNA requires active DNA methyltransferase enzymes.
  • Cells containing all three DNA methyltransferase enzymes (M1/3A/3B) were pulsed for 1 hr with BrdU and DNA collected after 1 hr from some of the cultures. The medium in the remaining cultures was changed to medium containing the indicated compounds during the chase period and DNA collected 24 hr after the beginning of the experiment.
  • BrdU-containing DNA was immunoprecipitated and the level of methylation in this DNA determined by quantitative MS-SNuPE analysis.
  • FIG 4B also shows that the maturation process was not blocked by drugs which inhibit histone deacetylase (Trichostatin A, "TSA”), DNA synthesis (Aphidicolin) or mitosis (Colcemid).
  • TSA histone deacetylase
  • Aphidicolin DNA synthesis
  • Colcemid mitosis
  • DNA collected from these two time points was immunoprecipitated to isolate BrdU-containing DNA, and the methylation status of individual molecules determined to assess the methylation density of these molecules as a function of time after synthesis as in Figure 1. Data are given as the percentage of individual molecules containing between 0 and 4 sites concurrently methylated on an individual molecule.
  • DNA collected immediately after the 1 hr pulse from M1/3A 3B cells showed that a considerable proportion (38%) of molecules contained none or only one of the four sites methylated and a relatively even distribution of methylation densities.
  • the pattern shifted substantially in the 24 hr chase period with most of the molecules now acquiring concurrent methylation at three or four sites.
  • Newly synthesized DNA in Ml cells had 40% of molecules with zero or one site methylated, 9% having two sites and 50% having three or four sites methylated. Again, this pattern shifted during the chase period, but the mode of the density distribution was two sites methylated per molecule as compared to the three in M1/3A/3B cells.
  • Example 5 Subcellular Localization of DNA Methyltransferases If the discontinuity observed in the kinetics of post-synthetic restoration of DNA methylation levels was indeed attributable to distinct methyltransferase activities, with Dnmtl mainly responsible for maintenance activity immediately following DNA synthesis and Dnmt3a and/or 3b being active later in the cell cycle, then Dnmt3a and/or 3b would not be expected to be as strongly associated with toroidal replication foci, as has been observed for Dnmtl.
  • the subcellular localization of the three methyltransferases was determined by the transfection of plasmids containing the three genes fused to green fluorescence protein (GFP) into mouse C3H 10T1/2 C18 cells. Cells were double stained with an antibody to PCNA labeled with Texas red and examined by confocal microscopy 48-72 hr after transfection. Cells transfected with DNMT-1 confirmed earlier studies that this protein often co-localized with PCNA, particularly in toroidal structures presumably representing replication machines at late S phase (Leonhardt et al., 1992; Chuang et al., 1997; Rountree et al, 2000).
  • Dnmt3a and 3b were localized almost entirely to the nucleus and were more diffusely present in nuclei containing these toroids.
  • Dnmt3b unlike Dnmt3a showed some co-localization with the PCNA-containing toroids.
  • Dnmt3b but not Dnmt3a may associate with replication machines.
  • EXAMPLE 6 Genetic scanning was used to identify sequences to investigate methylation patterns. The findings of Examples 1-5 (above) were extended. Specifically, a genome scanning approach was used to investigate the patterns of methylation in the various knockout ES cells in CpG poor and CpG rich regions to determine the roles of the enzymes in carrying out the bulk of methylation in mouse embryonic stem (ES) cells.
  • ES mouse embryonic stem
  • methylation levels of CpG-poor sequences were, in general, uniformly reduced in Dmntl -deficient cells. However, there was considerable variability among different regions in the efficiency with which DNA methylation was retained in Dnmt3a/3b-deficient cells indicating a sequence preference of the Dnmtl enzyme.
  • MS AP-PCR was used to fingerprint the methylation patterns in a panel of ES cells containing different combinations of DNA methyltransferases (Dnmtl (-/-) or Dnmt3a (-/- )/3b (-/-)).
  • the MS AP-PCR method allows for a methylation pattern to be easily obtained and relies on the differential susceptibilities of unmethylated and methylated CCGG sites to cutting by the enzyme Hpall giving a valid fingerprint of the methylation status of CpG islands.
  • the purpose and advantage of Ms AP-PCR was to perform a rapid and global screen of genome which would then allow identification of representative sequences for more detailed analysis. We initially focused on CpG poor regions of DNA in the current work, since these are the regions of DNA in which the majority of 5-methylcytosine is found.
  • Figure 7A shows an example of an analysis of DNA extracted from the various cell types using CpG poor primers to target regions of DNA not located in CpG islands.
  • the nomenclature used for the cells was selected to highlight which gene products were active in the cells rather than those which were absent.
  • Analysis of the fingerprints showed a uniform loss of methylation at most evaluable bands in cells which contained Dnmt3a and 3b only (M3A/3B), as compared to the other cell types examined (see lanes 2 and bands indicated by filled in arrows).
  • Class I sequences as those that had decreased methylation in M3A/3B cells (i.e., lacking Dnmtl), but close to normal methylation levels in Ml cells.
  • Class II sequences on the other hand showed loss of methylation in both Ml and M3A/3B cells.
  • Figure 8 shows the sequence properties of a selection of these two classes of fragments with respect to the occurrence of the CpG dinucleotides and the presence of repetitive elements.
  • Class I sequences tended to have fewer repetitive elements, and a slightly lower CpG density than Class II sequences.
  • this distinction was not absolute since there were no obvious differences with respect to these properties such as the fragments Cl-b and Cll-a, or Cl-a and Cll-b.
  • the 440-bp fragment Cl-f sequence (GenBand Accession number AK014019) showed extensive methylation of all four sites sequenced in the wild-type cells and extensive methylation also in the Ml cells but a marked decrease in M3A/3B cells on both the top and bottom strands, which had approximately equal methylation levels at the CpG sites in all cell types.
  • Analysis of the top strand of individual molecules in the M3A/3B cells showed a strong asymmetry of patterns in that 72% of molecules were completely unmethylated, 14% had only one site methylated, but 14% had multiple sites methylated on a single molecule.
  • Dnmt3a or 3b did not seem to be independently responsible for the methylation of these sites, since these sequences were also substantially demethylated in M3A/3B cells ( Figures 7 and 9). Furthermore, the effects of Dnmtl and Dnmt3a/3b did not appear to be merely additive ( Figure 9), as would be the case if both Dnmtl and Dnmt3a/3b were independent participants in the maintenance and de novo methylation of these sequences. Rather, methylation of these sequences appears to require cooperation both Dnmtl and Dnmt 3a/3b activities.
  • Dnmt3a and/or Dnmt3b are responsible for the compensating de novo methylation was provided by the fact that these enzymes could restore methylation to pretreatment levels following transient exposure of cells to 5-Aza-CdR, whereas Dnmtl could not.
  • Dnmtl by itself was also found herein to be incapable of restoring methylation of sequences that it had been able to maintain prior to the 5-Aza-CdR treatment, suggesting that its ability to de novo methylation is dependent on the presence of a critical level of preexisting methylation at CpG sites.
  • a New Assay for the Detection of Hemi-methylation One prediction of poor maintenance methylation of Class II sequences, offset by continuing de novo methylation is that the level of hemi-methylation at these sites (i.e., when only a single cytosine in the palindrome is methylated) should be substantially higher than in sequences with low levels of de novo methylation and very efficient maintenance methylation following each round of DNA replication. Testing of this prediction required the development of a way to measure levels of hemi-methylation at individual CpG dinucleotides.
  • the present invention provides such an invention as described herein under Examples 1-5 (see Figures 2 and 3). The assay was expanded to cover additional class I and class II genomic sequences.
  • Figure 10 shows the results of the hemi-methylation analysis of a Class I sequence (Cl-f) and of a Class II sequence (CII-d).
  • Cl-f Class I sequence
  • CII-d Class II sequence
  • the knockout cells were, therefore, exposed to 5-aza-CdR and followed the kinetics of remethylation of the Cl-f and CII-d fragments were followed to determine how the Dnmt's interacted to restore methylation levels after perturbation of the equilibrium in the ES cell types (Figure 11).
  • the results showed that methylation levels reached their minimum three days after drug treatment in wild type, and that the methylation of both fragments was restored to near pre-treatment levels by 14 days after treatment.
  • Figure 11 also shows that the combination of Dnmt3a with 3b (M3A/3B) was sufficient to result in the rapid remethylation of the CII-d but not the Cl-f fragment.
  • Dnmt3a and/or Dnmt3b methylation occur ed close to the time of DNA replication, while Dnmtl showed a substantial amount of delayed methylation
  • Methylation by Dnmt3a and/or Dnmt3b occurs close to the time of DNA replication, while Dnmtl shows a substantial amount of delayed methylation, extending beyond one hour post DNA synthesis.
  • this delay in maintenance methylation by Dnmtl was not responsible for the sequence-dependent variability in methylation levels in Dnmt3a/3b- deficient cells, since both types of sequences showed this maintenance methylation delay.
  • DNA synthesized during a 1-hr pulse of BrdU in wild-type (M1/3A/3B) cells was not methylated to its final level immediately after synthesis but that some further methylation occurred in the post-synthetic phase measured at 3.5 and 24 hr. However, this did not appear to be de novo methylation attributable to Dnmt3a and/or Dnmt3b, since M3A/3B cells did not show additional methylation after the first hour. Indeed, the delayed increase in methylation was also evident for the Cl-f sequence in Ml cells.
  • Dnmt3a and/or Dnmt3b occured immediately before or soon after DNA synthesis, while Dnmtl continued to display maintenance activity more than 1 hour post replication.
  • Dnmtl, Dnmt3a and 3b were used to show that DNA methylation in a CpG-poor transcribed sequence occurs in two phases with respect to DNA synthesis.
  • BrdU pulse chase experiments showed that Dnmtl functioned at or soon after replication to methylate the newly synthesized strand to approximately 72% of the equilibrium level, but leaves patches of hemimethylated DNA. The restoration of the pattern was significantly facilitated by
  • Dnmt3a and 3b which appear to be processive enzymes whose activities fill in the patches in addition to their roles as de novo methylases.
  • the present invention provides novel methods to measure hemi-methylation and to calculate methylation rates. These novel methods were used herein to conclude that Dnmt3a and 3b act mainly as maintenance methyltransferases to ensure the completeness of methylation of the genome in ES cells.
  • Dnmt3a and 3b act as de novo enzymes, and likely have more important roles in non-differentiating cells may be to assist Dnmtl in ensuring full maintenance of methylation patterns.
  • the methylation of a region of DNA occurred in two phases and therefore discontinuously (summarized in the model shown in Figure 6A, B and C).
  • FIGS 6A, B and C illustrate the proposed model for the interactivity between DNA methyltransferase enzymes in ES cells.
  • This figure represents a highly schematic interpretation of the data of the present invention.
  • the data is based on the analysis of four contiguous CpGs, and of one CpG dinucleotide in particular detail, the conclusions have been summarized using a hypothetical replication fork in which evenly spaced CpG dinucleotides are represented by circles. Black circles indicate methylated CpGs on the parent DNA strands, while grey circles represent newly-methylated CpGs on the daughter strand. Unmethylated CpGs are indicated by white circles.
  • PCNA is indicated by a ring.
  • DNA methyltransferases are indicated by rectangles (Dnmtl) or ovals (Dnmt3a and/or 3b).
  • Figure 6A depicts how methylation patterns are maintained in wild-type cells containing all three DNA methyltransferase enzymes. Most DNA synthesized in wild-type cells is likely immediately methylated after synthesis by Dnmtl which is presumably closely associated with the replication machinery through its interaction with PCNA. There are regions of DNA, in which patches of hemimethylation occur, and these appear to serve as substrates for the Dnmt3a and 3b enzymes The full copying of the pattern requires the concerted efforts of all three enzymes.
  • Figure 6B indicates how methylation is proposed to occur in cells lacking both
  • Dnmt3a and Dnmt3b enzymes Individual CpGs, or patches of CpGs missed by Dnmtl remain unmethylated, since Dnmt3a and Dnmt3b are lacking. This results in a higher incidence of unmethylated CpGs surviving to the next round of replication. Some of these CpGs are methylated in the next round of replication by Dnmtl, so that by 24 hours, there are relatively few large patches of unmethylated CpGs in Ml cells (see Figure 5).
  • Figure 6C shows the situation in M3A/3B cells, which lack Dnmtl.
  • a mosaic methylation pattern is present with patches of methylation and unmethylation and a substantial fraction of hemi-methylated sites. It is likely that in the absence of Dnmtl, Dnmt3a and/or 3b are capable of some copying of the existing methylation pattern, but are not particularly efficient.
  • the enzymes also seem capable of a de novo modification of both strands of DNA, thus resulting in a mosaic pattern of methylation within these cell types.
  • the first phase of methylation in which CpG sites are methylated to approximately 72% of their final levels, is most likely accomplished by Dnmtl, which has been shown to be associated with DNA replication machines by the elegant work of Leonhardt et al. (1992) and Rountree et al. (2000) and confirmed here.
  • the enzyme left gaps of unmethylated sites linked to each other as shown by the direct sequencing of BrdU pulse labeled DNA.
  • the size of these gaps or patches was not established, but they were at least 300 bp which is the distance between sites 1 and 4 in the fragment studied herein.
  • This DNA contained a substantial number of hemimethylated sites, which were converted into fully methylated sites during the post synthetic phase.
  • the delayed methylation was most likely accomplished by Dnmt3a/3b, which must therefore be acting as hemimethylases which seemed to be processive, yet not 100% efficient, enzymes.
  • the evidence for this processivity came from analysis of the M3A/3B cells which lacked Dnmtl in which 72% of the molecules analyzed had no methylation whereas 14% had more than one site methylated. Putting these two properties together probably assures the stability of the pattern. However, it is also possible that the enzymes are not processive but rather that the presence of methylation attracts further methylation to the region. Novel Hemi-methylation Assay.
  • the present invention provides a novel method for the quantitation of hemi-methylation, which requires complete digestion of DNA with HpaU before bisulfite treatment to ensure accuracy. This was accomplished by the use of an excess of enzyme, and replenishment of enzyme after 16 hr incubation.
  • the method is robust and has general utility in other experimental and applied applications.
  • the occurrence of a high percentage of hemimethylated sites in the M3 A/3B cells seemed, at first sight, to not fit with the suggestion that the Dmnt3a and 3b enzymes acted as hemi-methylases to fill in gaps in wild-type cells.
  • This apparent paradox could be explained if the overall level of cytosine methyltransferase activity in the M3A/3B cells was inadequate for effective methylation maintenance.
  • a pool of DNA molecules from a population of cells is comprised of many recently synthesized strands with DNA methylation levels at, or slightly below the average level for the entire population, as well as older DNA strands with higher levels of methylation.
  • the relative prevalence of a DNA strand in the population decreases as a function of the number of rounds of DNA replication since its initial synthesis. These "strands" may represent long patches, rather than entire molecules (chromosomes), since recombination and repair-associated DNA synthesis could disrupt their contiguity.
  • the model supports a mechanism, whereby DNA strands continue to become increasingly methylated as they age.
  • the rate calculations of the present invention should be viewed as weighted averages for the entire population of DNA strands of different age. Any de novo methylation that occurs on aging DNA strands could not be removed passively by DNA replication so that the only way to counteract this cumulative methylation would be to invoke active, enzymatic demethylation.
  • these aging strands represent an increasingly small fraction of the population, they could have biological consequences if they reach a threshold of DNA methylation that results in a phenotypic effect, such silencing a tumor-suppressor gene, leading to clonal outgrowth. This previously unrecognized phenomenon may explain, in part, the increase in CpG island hypermethylation observed in aging (Ahuja and Issa, 2000) and in cancer (Jones and Laird, 1999).
  • methylation properties of a non-CpG island region of DNA was measured. Although most genomic methylation occurs in such sequences, it will be important to see whether the same cooperation between the enzymes is evident with CpG islands where modification is more tightly associated with promoter silencing. In this regard, it was determined that methylation of repetitive sequences was not well maintained in ES cells lacking Dnmt3a and 3b, indicating that at least two methyltransferases are needed to ensure methylation of these sequences, unlike the fragment studied herein which was substantially methylated in Ml cells.
  • Examples 6-8 show that mouse ES cells with systematic gene knockouts for DNA methyltransferases (Dnmts) can be used to delineate the roles of Dnmtl and Dnmt3a and 3b in maintaining methylation patterns in the mouse genome.
  • Dnmtl alone was able to maintain methylation of most CpG-poor regions analyzed.
  • both Dnmtl and Dnmt3a and/or Dnmt3b were required for the methylation of a select class of sequences which included abundant murine LINE-1 promoters.
  • a novel hemi-methylation assay was used to show that even in wildtype cells these sequences contain high levels of hemi-methylated DNA, suggestive of poor maintenance methylation.
  • Dnmt3a and/or 3b could restore methylation of these sequences to prefreatment levels following transient exposure of cells to 5-Aza-CdR, whereas Dnmtl by itself could not.
  • ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmtl of these endogenous repetitive sequences.
  • the present results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.

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Abstract

La présente invention concerne un procédé qui permet de mesurer la fraction de molécules d'ADN qui est semi-méthylée au niveau d'un dinucléotide CpG spécifique, dans une séquence d'ADN particulière, dans un groupe de molécules d'ADN ayant des états de méthylation d'ADN mélangés au niveau dudit dinucléotide CpG; des procédés de calcul des taux de méthylation de novo et d'entretien par division cellulaire qui se produisent au niveau dudit dinucléotide CpG. Cette invention concerne également des trousses de méthylation destinées à être utilisées pour mesurer et quantifier la semi-méthylation et pour calculer les taux de méthylation de novo et d'entretien.
PCT/US2001/047141 2000-11-08 2001-11-08 Nouveau dosage permettant de detecter et de quantifier la semi-methylation Ceased WO2002038811A2 (fr)

Priority Applications (5)

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US10/416,075 US20040072197A1 (en) 2001-11-08 2001-11-08 Assay for the detection and quantitation of hemimethylation
AU2002233988A AU2002233988A1 (en) 2000-11-08 2001-11-08 Assay for the detection and quantitation of hemimethylation
CA002428245A CA2428245A1 (fr) 2000-11-08 2001-11-08 Nouveau dosage permettant de detecter et de quantifier la semi-methylation
EP01984994A EP1379685A2 (fr) 2000-11-08 2001-11-08 Nouveau dosage permettant de detecter et de quantifier la semi-methylation
JP2002541125A JP2004535757A (ja) 2000-11-08 2001-11-08 ヘミメチル化の検出及び定量のための新規アッセイ法

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US60/247,191 2000-11-08

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000842A3 (fr) * 2000-06-23 2003-06-19 Univ Chicago Methodes d'isolation d'adn centromere
DE10347399A1 (de) * 2003-10-09 2005-05-19 Epigenomics Ag Bisulfit-Umwandlung zum Nachweis von Cytosin-Methylierungen in DNA mittels optimierter Aufreinigung
WO2005033332A3 (fr) * 2003-09-30 2005-06-16 Epigenomics Ag Procede d'analyse de methylation d'adn
WO2005038051A3 (fr) * 2003-10-09 2005-06-16 Epigenomics Ag Transformation amelioree de bisulfite d'adn
EP1682677A4 (fr) * 2003-10-21 2007-12-19 Orion Genomics Llc Fragmentation enzymatique differentielle
EP1853724B1 (fr) * 2005-03-03 2009-10-21 Epigenomics AG Procédé d'analyse des méthylations de la cytosine dans l'adn
US7968295B2 (en) 2000-06-19 2011-06-28 Epigenomics Ag Bisulfite conversion of DNA

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6017704A (en) * 1996-06-03 2000-01-25 The Johns Hopkins University School Of Medicine Method of detection of methylated nucleic acid using agents which modify unmethylated cytosine and distinguishing modified methylated and non-methylated nucleic acids
US6251594B1 (en) * 1997-06-09 2001-06-26 Usc/Norris Comprehensive Cancer Ctr. Cancer diagnostic method based upon DNA methylation differences

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8257950B2 (en) 2000-06-19 2012-09-04 Epigenomics Ag Bisulfite conversion of DNA
US7968295B2 (en) 2000-06-19 2011-06-28 Epigenomics Ag Bisulfite conversion of DNA
US7132240B2 (en) 2000-06-23 2006-11-07 The University Of Chicago Use of methylated nucleic acid segments for isolating centromere DNA
WO2002000842A3 (fr) * 2000-06-23 2003-06-19 Univ Chicago Methodes d'isolation d'adn centromere
WO2005033332A3 (fr) * 2003-09-30 2005-06-16 Epigenomics Ag Procede d'analyse de methylation d'adn
DE10347399B4 (de) * 2003-10-09 2005-09-15 Epigenomics Ag Bisulfit-Umwandlung zum Nachweis von Cytosin-Methylierungen in DNA mittels optimierter Aufreinigung
AU2004282342B2 (en) * 2003-10-09 2009-05-14 Epigenomics Ag Improved bisulfite conversion of DNA
WO2005038051A3 (fr) * 2003-10-09 2005-06-16 Epigenomics Ag Transformation amelioree de bisulfite d'adn
DE10347399A1 (de) * 2003-10-09 2005-05-19 Epigenomics Ag Bisulfit-Umwandlung zum Nachweis von Cytosin-Methylierungen in DNA mittels optimierter Aufreinigung
EP1682677A4 (fr) * 2003-10-21 2007-12-19 Orion Genomics Llc Fragmentation enzymatique differentielle
US7901880B2 (en) 2003-10-21 2011-03-08 Orion Genomics Llc Differential enzymatic fragmentation
US7910296B2 (en) 2003-10-21 2011-03-22 Orion Genomics Llc Methods for quantitative determination of methylation density in a DNA locus
EP2292795A3 (fr) * 2003-10-21 2011-06-08 Orion Genomics, LLC Fragmentation enzymatique différentielle
EP2339024A1 (fr) * 2003-10-21 2011-06-29 Orion Genomics, LLC Procédés pour quantifier la densité de méthylation d'un site d'ADN
US8163485B2 (en) 2003-10-21 2012-04-24 Orion Genomics, Llc Differential enzymatic fragmentation
US8361719B2 (en) 2003-10-21 2013-01-29 Orion Genomics Llc Methods for quantitative determination of methylation density in a DNA locus
EP1853724B1 (fr) * 2005-03-03 2009-10-21 Epigenomics AG Procédé d'analyse des méthylations de la cytosine dans l'adn

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JP2004535757A (ja) 2004-12-02
WO2002038811A3 (fr) 2003-10-23
CA2428245A1 (fr) 2002-05-16
AU2002233988A1 (en) 2002-05-21

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